Glucansucrases are a type of glycosyltransferase (GTF) that belong to the glycoside hydrolase family 70 (GH70), as defined by the CAZy classification system (Cantarel B. L., et al., 2009, Nucleic Acids Res 37:D233-D238), and catalyze the transfer of D-glucopyranosyl units from sucrose to acceptor molecules to form α-glucan chains. Glucansucrases are capable of catalyzing the synthesis of several different α-glucosidic linkages that affect molecular mass, branching, and solubility of the polysaccharide. In general, α-glucans containing mostly α(1→6) linkages (e.g., dextran) are water-soluble, while those made primarily of α(1→3) linkages are water-insoluble. Sequences of α(1→6) linked glucose units tend to form a flexible chain which readily hydrates and dissolves in water, whereas sequences of α(1→3) linked glucose units tend to form extended ribbon-like helices which self-associate and are water-insoluble, similar to cellulose (Rees D A, et al., 1971, J Chem Soc 8:469-479; Yui et al., 2000, Biosci Biotechnol Biochem 64:52-60). The term “mutan” is often used to refer to the water-insoluble glucan produced by Streptococcus mutans and related bacteria (Guggenheim et al., 1970, Helv Odont Acta 14:89-108), and it has become well-established that “mutan” is a graft or block-type copolymer consisting of regions of dextran-like α(1→6) linkages sequences as well as sequences of α(1→3) linked regions. This block or graft copolymer structure is quite different from the highly water-soluble alternan, which has similar proportions of α(1→3) and α(1→6) linkages arranged in a regular, alternating fashion with no extended sequences of either linkage type (Côté, 2002, Chapter 13 in Biopolymers, Vol. 5. Polysaccharides I. Polysaccharides from Prokaryotes. E. J. Vandamme, S. DeBaets, A. Steinbiichel, Eds. Wiley-VCH, Weinheim, Germany. Pp. 323-350).
Whereas the utility of water-soluble dextran has been well-established (Leathers, 2002, Chapter 12 in Biopolymers, Vol. 5. Polysaccharides I. Polysaccharides from Prokaryotes. E. J. Vandamme, S. DeBaets, A. Steinbiichel, Eds. Wiley-VCH, Weiheim, Pp. 299-321), applications of the related water-insoluble glucans are much less developed. As such, there is a need to establish a range of related water-insoluble glucans with varying properties.
Three-dimensional structures and targeted modifications of glucansucrases have provided substantial information regarding the functionality of these enzymes (Vujicic-Zagar, et al., 2010, Proc Natl Acad Sci USA 107:21406-21411; Ito, et al., 2011, J Molec Biol 408:177-186), but the mechanisms that control the type of glycosidic linkage still remain unclear. Glucansucrases are often described based on amino acid alignments with other glucansucrase as having an N-terminal variable region, followed by a catalytic domain, and a C-terminal glucan-binding domain (Monchois, et al., 1999, J Bacteriol 181:2290-2292) however, structural analyses of Lactobacillus reuteri GTF180-AN and Streptococcus mutans GTF-SI show that these glucansucrase proteins that catalyze α(1→6)/α(1→3) linkages actually contain five domains (A, B, C; IV and V) that are formed through a U-shape configuration that involves two regions of the polypeptide for each domain, with the exception of domain C. The amino acid residues of the catalytic triad (aspartate-nucleophile; glutamate-acid/base; aspartate-transition state stabilizer) are located within a deep pocket of domain A, which has a (β/α)8 barrel structure (Vujicic-Zagar, id; Ito, id.).
It is thought that the amino acids following the transition state stabilizer determine the orientation of the acceptor molecules and therefore influence the type of glycosidic bond that is formed (Leemhuis et al., 2012, Biocatal Biotransform 30:366-376; Leemhuis et al., 2013, J Biotechnol 163:250-272). The fifth amino acid after the transition stabilizer is likely coupled with the +2 subsite that binds the acceptor molecule and is almost universally an aspartate or threonine among Streptococcus and a threonine with Leuconostoc species. In streptococci glucansucrase, aspartate is typically associated with insoluble glucan production, while threonine in this position usually results in synthesis of soluble glucan. Substituting D567T in S. mutans GtfB shifted production of soluble glucan from 0 to 24%. Conversely, T589D and T598E in GtfD went from 86 to 15% and 86 to 2%, respectively for each mutation (Shimamura, et al., 1994, J Bacteriol 176:4845-4850). Moreover, mutations of this equivalent position in S. downei GtfI influenced the structure of the glucan and size of oligosaccharide produced in studies using the catalytic core region in enzymatic assays (Monchois, et al., 2000, Appl Environ Microbiol 66:1923-1927).
Recently identified is a glucansucrase, DsrI, from the type strain of L. mesenteroides (NRRL B-1118) that produces a water-insoluble glucan containing approximately 44% α(1→3), 29% α(1→6), 15% terminal non-reducing end residues, and 9% branching through 1,3,6-trisubstituted α-D-glucopyranosyl units (Côté et al., 2012, Appl Microbiol Biotechnol 93:2387-2394). The linkage types apparently occur in discrete sequence domains, with linear segments of α(1→3) linkages interspersed with or grafted onto segments of α(1→6)-linked regions. There is no evidence for an alternan-like structure in the insoluble glucan. This enzyme contains a threonine in position 654 like most other Leuconostoc glucansucrases, but its product is unique due to the high percentage of α(1→3) linkages and water-insolubility. Given this property, there is a need to further scrutinize the amino acid at the 654 position to determine whether the catalytic properties of the enzyme and synthesize glucans with different structures can be modified.